Process for the purification of factor VIII and factor VIII obtained by said process

Abstract

A method for the purification of Factor VIII from human plasma is described, wherein a solution comprising Factor VIII is purified by using ion exchange chromatographic columns. Factor VIII obtained by said method is also described.

We claim: 
     
       1. Process for the purification of Factor VIII from human plasma comprising passing a solution containing Factor VIII through an ion exchange chromatographic column using both as an equilibrating buffer and as an eluent for adsorbed Factor VIII, a high ionic strength saline solution at an acid pH of from 6.4 to 6.8 and eluting said adsorbed Factor VIII with said high ionic strength saline solution and collecting a recovered eluate in presence of stabilizers and optionally of an antiprotease. 
     
     
       2. Process according to claim 1, wherein said chromatographic column contains an anion exchange resin. 
     
     
       3. Process according to claim 2, wherein the anion exchange resin comprises quaternary amino groups. 
     
     
       4. Process according to claim 1, wherein the equilibrating buffer consists of 10-30 mM Tris, 150-300 mM NaCl, 5-15 mM CaCl 2  and pH is from 6.4 to 6.8. 
     
     
       5. Process according to claim 4, wherein the equilibrating buffer consists of 20 mM Tris, 250 mM NaCl, 10 mM CaCl 2  and pH is 6.6. 
     
     
       6. Process according to claim 1 wherein the eluent for Factor VIII consists of 10-30 mM Tris, 450-650 mM NaCl, 5-15 mM CaCl 2  and pH is from 6.4 and 6.8. 
     
     
       7. Process according to claim 6, wherein the eluent for Factor VIII is 20 mM Tris, 550 mM NaCl, 10 mM CaCl 2  and pH is 6.6. 
     
     
       8. Process according to claim 1 wherein the eluted Factor VIII is stabilized with:   ______________________________________                                    
Albumin 5%      0.2-1.25 mg/ml eluate,                                    
heparin         0.02-0.125 U.I. g/ml eluate,                              
PEG 4000        0.006 mg/ml eluate                                        
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     and optionally an antiprotease.   
     
     
       9. Process according to claim 8, wherein lysine or histidine are used as antiprotease. 
     
     
       10. Process according to claim 9, wherein the antiprotease is used in concentration 10 mM. 
     
     
       11. Process for purification of Factor VIII from human plasma comprising: A) thawing human fresh frozen plasma with stirring, permitting a cryoprecipitate to form and collecting the cryoprecipitate by centrifugation;   B) solubilizing the cryoprecipitate in distilled heparinized water to form a solution and, treating the solution with an aluminum hydroxide suspension and then with PEG 4000, adjusting the pH with acetic acid 1N to a value 6.4-6.6 and cooling the solution thereafter to 9°-11° C.; removing by centrifugation the proteins adsorbed upon aluminum hydroxide and those precipitated leaving a supernatant;   C) passing said supernatant obtained in the previous stage through a chromatographic column containing an anion exchange resin by using an equilibrating buffer which consists of 10-30 mM Tris, 150-300 mM NaCl, 1-15 mM CaCl 2  and pH 6.4 to 6.8; then washing the resin with the aforementioned buffer in order to eliminate the undesired proteins and then recovering the factor von Willebrand and Factor VIII:C by eluting with an eluent buffer consisting of 10-30 mM Tris, 450-650 mM NaCl, 5-15 mM CaCl 2  and pH 6.4 to 6.8; stabilizing the so recovered Factor VIII:C with:   ______________________________________                                    
Albumin 5%      0.2-1.25 mg/ml eluate,                                    
heparin         0.02-0.125 U.I. g/ml eluate,                              
PEG 4000        0.006 mg/ml eluate                                        
______________________________________                                    
     concentrating, diafiltrating and further concentrating the obtained eluate solution up to a final volume equal to 174  starting volume;     D) pasteurizing, concentrating and dispensing the Factor VIII:C into vials and lyophilizing the Factor VIII:C.   
     
     
       12. Process according to claim 11, wherein the equilibrating buffer consists of 20 mM Tris, 250 mM NaCl, 10 mM CaCl 2  and pH is 6.6 
     
     
       13. Process according to claim 11, wherein the eluent buffer for Factor VIII is 20 mM Tris, 550 mM NaCl, 10 mM CaCl 2  and pH is 6.6. 
     
     
       14. Process for the purification of Factor VIII from human plasma comprising passing a solution containing Factor VIII through an ion exchange chromatographic column using as equilibrating buffer a high ionic strength saline solution consisting of 10-30 mM Tris, 150-300 mM NaCl, 5-15 mM CaCl 2  at pH from 6.4 to 6.8 and eluting an adsorbed Factor VIII with a high ionic strength saline solution consisting of 10-30 mM Tris, 450-650 mM NaCl, 5-15 mM CaCl 2  at the above said pH and collecting a recovered eluate in presence of stabilizers and optionally of an antiprotease. 
     
     
       15. Process according to claim 14, wherein the equilibrating buffer consists of 20 mM Tris, 250 mM NaCl, 10 mM CaCl 2  and pH is 6.6. 
     
     
       16. Process according to claim 14, wherein the eluent for Factor VIII is 20 mM Tris, 550 mM NaCl, 10 mM CaCl 2  and pH is 6.6. 
     
     
       17. Process according to claim 16, wherein said chromatographic column contains an anion exchange resin. 
     
     
       18. Process according to claim 17, wherein the anion exchange resin comprises quaternary amino groups. 
     
     
       19. Process according to claim 14, wherein the eluent Factor VIII is stabilized with:   ______________________________________                                    
Albumin 5%      0.2-1.25 mg/ml eluate.                                    
heparin         0.02-0.125 U.I. g/ml eluate.                              
PEG 4000        0.006 mg/ml eluate                                        
______________________________________                                    
     and optionally an antiprotease.   
     
     
       20. Process according to claim 19, wherein lysine or histidine are used as antiprotease. 
     
     
       21. Process according to claim 20, wherein the antiprotease is used in concentration 10 mM.